The present invention pertains to a process of the purification of a growth factor protein employing chromatography.
The purification of proteins from sources of natural origin is a challenge as the protein of interest is often only present in trace amounts and accompanied by other biopolymers such as lipids, proteins, or even cell fragments. Moreover the proteins of interest are mostly associated with a biological function which is often lost during process steps for its purification.
The arsenal of methods for purifying biopolymers such as proteins is large. Besides precipitation methods chromatographic methods on various kinds of materials are known. Frequently the materials are modified with chemical moieties such as organic ions, cations such as protonated amines or partially or completely alkylated amines. Such materials are used as anion exchangers. But also cation exchangers can be used for purification methods depending on the physical properties of the protein of interest such as shape, molecular weight and in particular its charge. Alternatively or in combination affinity chromatography is employed.
It is known in prior art that one disadvantage with traditional ion exchange chromatography resins (as for example SP-, CM-, Q- or DEAE SEPHAROSE® FF ion exchange chromatography resins) is that the binding of a protein to the resin only can be performed within relatively low salt concentration (conductivity, osmolality etc.), typically in the range of 0.01-0.15M of salt (NaCl etc.) concentration. In certain applications there would be a demand to be able to use the relatively mild purification conditions a ion exchange chromatography step exerts towards the proteins, also directly (without further dilution) to a chromatography resin at somewhat increased ionic strength. An increased ionic strength can be of significant advantage for the protein stability in a protein solution; especially in a crude protein preparation like in the harvest of recombinant produced protein products or in plasma derived products where potential proteases are present in the solution which can affect the target protein negatively. As proteases often work best at physiological conditions (like is the case in most cell systems), i.e approximately pH 7 and a salt concentration of approximately 0.15M.
Proteases could be inhibited by changing the work-up conditions eg by addition of salt and/or change of the pH, however, both these parameters are critical for the performance of a conventional ionic chromatography step and thus often impossible to use in their combination. There is a need to provide a purification method in the course of which conditions to minimise the effects of proteases can be employed.
WO-A2-2008/073620 discloses a manufacturing method for polypeptides that are produced in insect cells using a baculoviral expression system. In one example, the insect cell culture is supplemented with a lipid mixture immediately prior to infection (e.g., one hour prior to infection). The polypeptides are isolated from the insect cell culture using a method that employs anion exchange or mixed-mode chromatography early in the purification process. This process step is useful to remove insect-cell derived endoglycanases and proteases and thus reduces the loss of desired polypeptide due to enzymatic degradation. In another example, mixed-mode chromatography is combined with dye-ligand affinity chromatography in a continuous-flow manner to allow for rapid processing of the insect-cell culture liquid and capture of the polypeptide. In yet another example, a polypeptide is isolated from an insect cell culture liquid using a process that combines hollow fiber filtration, mixed-mode chromatography and dye-ligand affinity in a single unit Operation producing a polypeptide solution that is essentially free of endoglycanase and proteolytic activities. In a further example, the isolated polypeptides are glycopeptides having an insect specific glycosylation pattern, which are optionally conjugated to a modifying group, such as a polymer (e.g., PEG) using a glycosyltransferase and a modified nucleotide sugar.
WO-A2-2009/063069 discloses a process for purifying peptides, in particular but not exclusively, to a process for removing endotoxins from a peptide solution, to a kit comprising reagents for said process and to the purified peptide obtained by said process.
Dasari Venkata Krishna Rao et al. discloses a purification method employing a process control-strategy developed for improving the yield of rhG-CSF (recombinant human granolocyte colony-stimulating factor). A purity of ≥99% with an overall yield of 2.18 g/l was achieved in the present study. Analysis of the product during purification indicated that detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs (host cell proteins) without removing nucleic acid. Cysteine concentration was a key parameter in protein refolding. The bed height and HETP (height equivalent theoretical plates) value in the SEC (size-exclusion chromatography) column was evaluated and its impact on the resolution was studied. Formulation during SEC was found to be crucial for increasing the product yields with saving of time and process costs.
Quan Bai et al. studies the renaturation and purification of recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF) expressed in Escherichia coli with strong anion-exchange chromatography (SAX). The effects of pH values, ratios of concentrations of GSH/GSSG, and urea concentrations in the mobile phase on the renaturation and purification of rhGM-CSF with SAX were investigated, respectively. The results show that the above three factors have remarkable influences on the efficiency of renaturation and mass recovery of rhGM-CSF. The addition of GSH/GSSG in the mobile phase can improve the formation of correct disulfide bonds in rhGM-CSF so that its renaturation yield increases. In addition, to enhance the mass recovery of rhGM-CSF with SAX, the low concentration of urea was added in the mobile phase to prevent denatured protein aggregation. Under the optimal conditions, rhGM-CSF was renatured with simultaneous purification on SAX column within 30 min only by one step.
Shelly A. Pizarro reports about a vascular endothelial growth factor (VEGF165) which is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. The process described in this report involves a bacterial expression system capable of producing approximately 9 g of rh VEGF per liter of broth and a downstream purification process of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in perisplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30±6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry. N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays.
Kimberly A. Kaleas discloses that mixed-mode chromatography resins are gaining popularity as purification tools for challenging feedstocks, and discloses the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on CAPTO® MMC from an alkaline feedstock. CAPTO® MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen bonding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. It is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded and pH 9.8 in the presence of urea and redox agents. The unique mixed mode characteristics of CAPTO® MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, L-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the CAPTO® MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.
One object of the invention was to avoid the drawbacks of the purification processes of a growth factor protein of prior art by providing a novel process. According to the invention the object is accomplished by a process of purifying a growth factor protein selected from the group consisting of Colony Stimulating Factor (CSF) such as G-CSF (Granulocyte Colony Stimulating Factor) or granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), Hepatocyte growth factor, Epidermal growth factor and fibroblast growth factor (acid) in a purification sequence employing chromatography wherein                at least one chromatography is performed using a multimodal resin        the Growth Factor Protein binds to the multimodal resin at a pH between 4 to 6.2, and        the Growth Factor Protein is eluting from the multimodal resin at a pH >6.3.        
The invention provides a process in which advantageously the effects of proteases can be minimised. Making it possible to add salt and/or change the pH in crude protein sample with potential proteases present which could degrade the target protein and to process the protein solution without any further measures and bind the target protein to a mixed mode chromatography resin and thus providing a optimized step for concentration and purification of the target protein in a crude sample, making it suitable for further purification downstream using a specific affinity chromatography step directed towards the target protein, with reduced protease and/or DNA content during the downstream processing. This is of specific importance, to avoid degradation of the target protein during purification, making the combination of multimodal chromatography as a capture step in a crude protein solution.
In one embodiment, the chromatography on multimodal resins is combined with a yeast derived affinity ligand chromatography step. The chromatographic step employing the yeast derived affinity ligand, is especially suitable for the purification of the target protein in high yield and an unchanged molecule integrity (degradation etc.).
The Growth Factor Protein is a Colony Stimulating Factor (CSF) such as G-CSF (Granulocyte Colony Stimulating Factor). This is a member of the hemopoietic regulatory glycoproteins which are involved in the growth and differentiation of hemopoietic cells from stem cells. Growth Factor Proteins are granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), Hepatocyte growth factor, Epidermal growth factor and fibroblast growth factor (acid). The Growth Factor Proteins all show an IP≤6. In a further embodiment of the invention the multimodal resin comprises moieties bound to a matrix and the moieties are able to interact with the Growth Factor Protein in a mixture by ionic interactions and other types of interactions such as hydrogen bonding, hydrophobic and thiophilic interactions.
In a further embodiment of the invention the affinity ligand is a yeast derived Fab fragment directed towards the growth factor protein.
In a further embodiment of the invention the multimodal resin step is processed to capture the Growth Factor Protein from a crude protein solution whereafter processing the resulting multimodal chromatography resin eluate to the yeast derived affinity ligand chromatography step and after elution of the Growth Factor protein from said affinity chromatography step, exerting a purity of more than approximately 90% in relation to proteins and DNA.
In another further embodiment of the invention the multimodal resin step and the yeast derived affinity ligand chromatography step is combined with other chromatography purification step to exert a purity of more than 99% in the final Growth Factor Protein product.
In still another embodiment of the invention the mixture comprising the Growth Factor Protein is a solution.
In yet another embodiment of the invention the Growth Factor Protein is a recombinant Growth Factor Protein.
In yet another embodiment of the invention the Growth Factor Protein is in a crude protein solution including potentially proteases which can degrade the product.
In another embodiment the Growth Factor Protein is eluted by a pH change >pH 6.3.
In a further embodiment of the invention the elution is performed with an elution agent comprising an amino acid having a basic side chain and/or high ionic strength. Alternatively or in combination the elution can also be performed by a pH change. The pH change is performed by adjusting the pH of the elution buffer with for example sodium hydroxide or acetic acid to desired pH and thereafter applying the buffer to the multimodal resin and the ionic strength adjustment can be performed by adding salt, in the elution buffer composition before applying to the multimodal resin, for example salts included in the Hofmeister series, for example sodium chloride and potassium chloride.
According to the invention the concentration of the elution agent is in particular in the range of from about 0.1M to about 2 M According to another embodiment of the invention the Growth Factor Protein binds to the multimodal resin at about pH 6.0 whereas the Growth Factor Protein is eluted from the multimodal resin at a pH about 6.5 or higher in particular at about pH 7.0.
In a further embodiment of the invention a buffering substance is used comprising preferably at least one of the substances selected from the group consisting of sodium citrate, histidine, 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonic acid (HEPES), 2-(N-Morpholino)ethane sulfonic acid (MES), Tris base and sodium acetate in particular in a range of about pH 4 to about pH 8. In the process of the invention one non-ionic detergent can be present in any of the buffers used, which non-ionic detergent is in particular selected from the group consisting of Polysorbates (Polysorbate 20, 40, 60, 80) and PLURONIC® F68.
In a further embodiment of the process of the invention the amino acid can be selected from the group of amino acid having a basic side chain including arginine, lysine and histidine; the organic salts can be selected from the group of KCl and NaCl
In another embodiment of the invention a wash step is performed at a pH in the range of about pH 4 to about pH 6, before eluting the Growth Factor Protein from the multi modal resin, characterised that the wash buffer includes washing agents comprising an amino acid having a basic side chain and/or high ionic strength, the ionic strength adjustment can be performed by adding salt, in the wash buffer composition before applying to the multimodal resin, for example salts included in the Hofmeister series, for example sodium chloride and potassium chloride
According to the invention the concentration of the washing agent is in particular in the range of from about 0.1M to about 2M
It can be advantageous to apply the washing buffer to the multimodal resin, to wash away contaminants (proteases, DNA etc.) and retain the Growth Factor Protein, before the Growth Factor Protein is released.
Particularly, the concentration of the amino acid which is positively charged at a pH 6-8 is present in an amount of up to 2M in the wash buffer at a pH of <6.3. Typically, the amount of arginine is in the range of 0.1-1.0M, in particular 0.5M in the wash buffer.
In the elution buffer with a pH≥6.3 the amount of arginine is typically in the range of 0.1 to 2M, in particular 0.5M.
In the elution buffer with a pH≥6.3, sodium chloride is included in a range of 0.1-2.0M, in particular in a range from 0.1 to 1M.
In the wash buffer with a pH≤6.3, sodium chloride is included in a range of 0.1-2.0M, in particular in a range from 0.1 to 1M.
The amount of non-ionic detergent is typically in the range of 0.001 to 1%, in particular in the buffers for multimodal chromatography 0.02%.
The multimodal chromatography resin which can be employed according to the invention may contain at least one of the following moieties:                a. a positively charged N-Benzyl-N-methyl ethanolamine ligand,        b. a negatively charged 2-(benzoylamino) butanoic acid ligand,        c. a phenylpropyl ligand,        d. a N-hexyl ligand,        e. a 4-Mercapto-Ethyl-Pyridine ligand,        f. a 3-((3-methyl-5-((tetrahydrofuran-2-ylmethyl)-amino)-phenyl)-amino)-benzoic acid ligand or combinations thereof.        
In particular, a multimodal chromatography resin for use according to the present invention is selected from the following commercially available resins HEP Hypercel™; PPA Hypercel™; CAPTO® Adhere™; CAPTO® MMC™; MEP Hypercel™.
In another embodiment of the present invention the purification sequence may further comprise pathogen removal/inactivation steps comprising a chemically based inactivation step, a size based removal step, chromatography steps or combinations thereof which steps are based on different physiological properties directed to the pathogen to be removed.
In a particular embodiment the process of the invention the purification sequence further comprises the following steps:                1. a cation multimodal resin such as CAPTO® MMC;        2. a chemically based inactivation step for enveloped viruses in particular the solvent/detergent-inactivation employing tri-n-butyl phosphate and Triton X-100 as disclosed in EP-A-131 740;        3. an affinity resin based on a ligand expressed in yeast;        4. a cation exchanger such as SP SEPHAROSE® or Resource S;        5. a pathogen filtration removal step with a mean pore sized of about 20 nm such as PLANOVA® 20N;        6. a buffer exchange and/or concentrating step such as ultra filtration with an approximate cut off of 1-5 kDa;        7. a size exclusion chromatography resin such as SUPERDEX® 75.        
The invention is further described by the following non-limiting examples which have been exemplified by G-CSF purification.